Proton‐Gradient‐Driven Sensitivity Enhancement of Liposome‐Encapsulated Supramolecular Chemosensors

Abstract An overarching challenge in the development of supramolecular sensor systems is to enhance their sensitivity, which commonly involves the synthesis of refined receptors with increased affinity to the analyte. We show that a dramatic sensitivity increase by 1–2 orders of magnitude can be achieved by encapsulating supramolecular chemosensors inside liposomes and exposing them to a pH gradient across the lipid bilayer membrane. This causes an imbalance of the influx and efflux rates of basic and acidic analytes leading to a significantly increased concentration of the analyte in the liposome interior. The utility of our liposome‐enhanced sensors was demonstrated with various host–dye reporter pairs and sensing mechanisms, and we could easily increase the sensitivity towards multiple biologically relevant analytes, including the neurotransmitters serotonin and tryptamine.


Instruments
Dynamic light scattering (DLS) measurements were performed with a Zetasizer Nano from Malvern Instruments and fluorescence measurements were performed in quartz glass cuvettes with a Varian Cary Eclipse spectrofluorometer or a Jasco FP-8300 spectrofluorometer equipped with temperature-controlled stirrers.

Buffer Preparations
All buffers were prepared by dissolving the acid form of the buffer and addition of NaOH to adjust the desired pH. For example, the sodium citrate buffers were prepared from citric acid, the 100 mM NaH2PO4, pH 7.5 buffer from sodium dihydrogen phosphate and the 100 mM Na2HPO4, pH 10.8 buffer from disodium hydrogen phosphate with subsequent addition of NaOH to adjust the pH.

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Liposome Preparations CB8/MDAP liposomes (POPC/POPS⊃CB8/MDAP). 100 µL 25 mg/ml POPC and 33 µL 10 mg/ml POPS in chloroform were mixed in a 5-mL round bottom flask and purged with nitrogen to obtain a thin lipid film. The film was dried overnight under high vacuum and rehydrated with 1 mL rehydration buffer (0.5 mM CB8 and 0.55 mM MDAP in either 100 mM Hepes, pH 7.5 or 100 mM sodium citrate, pH 3.5). After gentle agitation at ambient temperature for 30 minutes, the liposome suspensions were subjected to 15 freeze-thaw cycles. The external buffer was subsequently exchanged by size exclusion chromatography (NAP 25) with 100 mM Na2HPO4, pH 10.8.
CB7/BE liposomes (POPC/POPS⊃CB7/BE). 100 µL 25 mg/ml POPC and 33 µL 10 mg/ml POPS in chloroform were mixed in a 5-mL round bottom flask and purged with nitrogen to obtain a thin lipid film. The film was dried overnight under high vacuum and rehydrated with 1 mL 100 mM sodium citrate, 0.3 mM CB7, 0.5 mM BE, pH 3.5. After gentle agitation at ambient temperature for 30 minutes, the liposome suspensions were subjected to 15 freezethaw cycles. The external buffer was subsequently exchanged by size exclusion chromatography (NAP 25) with 100 mM Na2HPO4, pH 10.8.
CB7/PLM liposomes (POPC/POPS⊃CB7/PLM). 100 µL 25 mg/ml POPC and 33 µL 10 mg/ml POPS in chloroform were mixed in a 5-mL round bottom flask and purged with nitrogen to obtain a thin lipid film. The film was dried overnight under high vacuum and rehydrated with 1 mL rehydration buffer (1 mM CB7 and 1 mM PLM in either 100 mM NaH2PO4, pH 7.5 or 100 mM sodium citrate, pH 3.5). After gentle agitation at ambient temperature for 30 minutes, the liposome suspensions were subjected to 15 freeze-thaw cycles. The external buffer was subsequently exchanged by size exclusion chromatography (NAP 25) with 100 mM Na2HPO4, pH 10.8.

HP-β-CD/BE liposomes (POPC/POPS⊃HP-β-CD/BE).
200 µL 25 mg/ml POPC and 66 µL 10 mg/ml POPS in chloroform were mixed in a 5-mL round bottom flask and purged with nitrogen to obtain a thin lipid film. The film was dried overnight under high vacuum and rehydrated with 1 mL rehydration buffer (20 mM HP-β-CD and 1 mM BE in either 100 mM NaH2PO4, pH 7.5, 100 mM sodium citrate, pH 3.5, or 100 mM Na2HPO4, pH 10.8). After gentle agitation at ambient temperature for 30 minutes, the liposome suspensions were subjected to 15 freeze-thaw cycles. The external buffer was subsequently exchanged by size exclusion chromatography (NAP 25) with either 100 mM Na2HPO4, pH 10.8 or 100 mM sodium citrate, pH 3.0.
HPTS liposomes (POPC/POPS⊃HPTS). 200 µL 25 mg/ml POPC 66 µL 10 mg/ml POPS in chloroform were mixed in a 5-mL round bottom flask and purged with nitrogen to obtain a thin lipid film. The film was dried overnight under high vacuum and rehydrated with 1 mL rehydration buffer (1 mM HPTS 100 mM NaH2PO4, pH 7.2). After gentle agitation at ambient temperature for 30 minutes, the liposome suspensions were subjected to 15 freeze-thaw cycles. The external buffer was subsequently exchanged by size exclusion chromatography (NAP 25) with 100 mM Na2HPO4, pH 10.8.
The concentrations of the phospholipids in the resulting liposome stock solutions was determined by our NMR method [3] and the size of the liposomes was determined by DLS. The total phospholipid concentrations were ca. 27 µM for experiments with the CB8/MDAP, CB7/BE, and CB7/PLM reporter pairs and ca. 55 µM for the HP-β-CD/BE reporter pair. For all liposome preparations, an unimodal size distribution was observed (Fig. S1) and the size of all liposomes was in the range of ca. 150-200 nm (Table S1).  S-5

Liposome Stability
The experiments were usually performed within four days after liposome preparation. During that time, no alterations in the liposome size or in the performance of the liposomeencapsulated reporter pairs was noted. Also, the fluorescence intensity of the liposomes did not change significantly (Fig. S2).

Titrations in Homogeneous Solution
Conventional fluorescence titrations were performed and the data was analyzed as previously described. [4]

Titrations with Liposome-Encapsulated Reporter Pairs
Titrations with the liposome-encapsulated reporter pairs were performed by measuring the time-dependent fluorescence changes of the reporter pairs at suitable excitation and emission wavelengths (CB8/MDAP: λex = 338 nm, λem = 423 nm; CB7/BE: λex = 420 nm, λem = 495 nm; CB7/PLM: λex = 425 nm, λem = 495 nm; HP-β-CD/BE: λex = 420 nm, λem = 540 nm). Therefore, 20 µL of reporter pair-encapsulated liposomes were diluted with 1980 µL 100 mM Na2HPO4, pH 10.8 for basic analytes or 100 mM sodium citrate, pH 3.0 for acidic analytes in fluorescence quartz glass cuvettes. Fluorescence was then monitored continuously during successive addition of the analytes, whereas the sample was allowed to fully equilibrate inside and outside concentrations after each addition as indicated by a constant fluorescence intensity. The total volume of added analyte stock solution did not exceed 100 µL (<5%), such that fluorescence intensity changes originating from dilution were not corrected. The constant fluorescence intensity after equilibration was then plotted against the total concentration of added analyte and the data was analyzed as previously described (assuming homogeneous solution conditions, see next paragraph).

Comparability of Measurements in Liposomes and Homogeneous Solution
To afford a reliable comparison of the fluorescence changes and the resulting apparent and true binding constants of the analytes with liposome-encapsulated reporter pairs and reporter pairs in homogeneous solution, it is most desirable that the reporter pair concentrations are below the dissociation constant, Kd, of the analyte. In this case, the shape of the fluorescence response curves become independent of the reporter pair concentrations, whereas at reporter pair concentrations much above the Kd of the analyte, the response of the sensor would depend on the reporter pair concentrations. As an extreme example, quantitative binding results at concentrations much above the Kd, such that full displacement (100% response) occurs when [host]tot = [analyte]tot. It was thus ensured that the concentrations of the reporter pair concentrations were below the Kd of the analyte in solution for all investigated combinations (except for 2-phenethylamine with CB7/PLM at pH 3.5). Additionally, the concentrations of the liposome-encapsulated reporter pairs were always below the reporter pair concentrations in homogeneous solution to ensure that an overlooked dependence on the reporter pair concentration would rather lead to a decreased than to increased sensitivity of the liposomeencapsulated reporter pairs.

Apparent Binding Constants
Apparent binding constants in 5% blood serum were measured by adding 20 µL of reporter pair-encapsulated liposomes to 1880 µL of the respective buffer (see above), 100 µL blood serum and successive addition of varying amounts of analyte. After each addition, the sample was allowed to fully equilibrate inside and outside concentrations and the constant fluorescence intensity was plotted and analyzed as described above. In homogeneous solution, 100 µL were added to 1900 µL buffer containing the reporter pair and titrations were conducted and analyzed as described above.

LOD Determinations
Spiked blood serum samples were prepared by adding known amounts of the analyte from a highly concentrated stock solution to human blood serum such that dilution effects of the blood serum can be neglected. 100 µL of the spiked blood serum samples were added to 1900 µL reporter pair (either liposome-encapsulated or in solution) and the fluorescence was recorded until a constant value was reached. The constant fluorescence intensity was then plotted against the analyte concentration the spiked blood serum samples. LOD values were then calculated from the slope and standard deviation of the linearly fitted line.       [14] Fe N(CH3)2  Figure S33. Absorption spectrum of 5% blood serum in 100 mM sodium phosphate buffer, pH 10.8.